Salmonellosis continues to be a major health problem in the United States and the rest of the world. Penetration of intestinal epithelial cells is a common feature of all Salmonella species. Despite the central importance of this event in the pathogenicity of these organisms, little is known about its molecular bases. The broad objective of this study is to understand the molecular mechanisms of Salmonella invasion of non- phagocytic cells and to identify all the biochemical components involved in this interaction. The principal investigator has recently cloned a group of genes (invA, B, C and D) that allow S. typhimurium to penetrate tissue culture cells. The functional study of these genes by a combination of biochemical, tissue culture and genetic techniques will be the main focus of this research. Mutated inv genes will be introduced into wild-type S. typhimurium to determine their specific role in invasion to cultured epithelial cells. The localization of the inv gene products in the bacterial cell as well as their ability to bind to eukaryotic cells will be examined. Fusions to reporter genes will be employed to examine the regulation of expression of the inv genes in response to exposure to tissue culture cells as well as to other environmental influences. The role of DNA supercoiling in this context will be particularly examined using in vivo and in vitro techniques. Mutations that affect expression of the inv genes will be isolated to characterize regulatory loci. Additional pathways of Salmonella invasion to tissue culture cells will be sought by isolating invasion defective mutants and characterizing the mutated loci. The studies proposed will increase the understanding of the molecular bases of Salmonella invasion to non-phagocytic cells. This knowledge will in turn facilitate the developing of new strategies to prevent and treat diseases caused by these and other invasive bacteria.